Somatic Mutations Associated with IgV_H4-34 FR1 Region Unmutated QW and Avy Motifs in DLBCL Patients

There is increasing evidence for antigen-driven B-cell receptor (BCR) signalling in diffuse large B-cell lymphoma (DLBCL) and other lymphoid malignancies. This includes antigens from infections e.g.Helicobacter pyloriand Hepatitis C virus, but it is theorised that self-antigens may play a major role in some cases of lymphoid malignancy. IgV_H4-34 demonstrates intrinsic autoreactivity to self-antigens on red cells, which appears to be largely mediated by two motifs within the first framework region (FR1); Q⁶W⁷ and A²⁴V²⁵Y²⁶. These motifs work together to for a hydrophobic patch which determines red cell antigen binding and are frequently mutated away from self-reactivity in normal B cells. IgV_H4-34 has been reported to be over-represented in DLBCL compared with expression in normal B cells.

We therefore sought to identify IgV_H4-34 DLBCL cases from a local cohort and to screen them for Q⁶W⁷ and A²⁴V²⁵Y²⁶ motifs expecting them to be less frequently mutated in DLBCL compared with normal B cells.We also aimed to screen V4-34 cases for associated somatic mutations in other genes using high-throughput sequencing.

DLBCL patient samples were obtained via the Haematology Research Tissue Bank (HRTB) in Canberra, Australia, and the Victoria Cancer BioBank. Forty-eight Formalin-Fixed, Paraffin-Embedded (FFPE) samples and 26 fresh frozen samples were screened. All samples were collected at the time of diagnosis. Patients were treated with standard chemoimmunotherapy approaches.

IgV_H 4-34 gene sequences were determined using an IgV_H4 family-specific leader primer in combination with a JH consensus reverse primer. The IgV_H region was then sequenced using Sanger sequencing. Sequences were analyzed using the IgBLAST database (National Centre for Biotechnology Information).

DNA extracted from FFPE samples generally proved to have low concentration and fragmented DNA. Only 1 IgV_H4-34 sequence was obtained from FFPE tissue.

Five samples sequenced from fresh tissue were identified as using IgV_H4-34. Using Hans criteria, it was possible to classify 3 of the 6 cases as germinal center (GC) and 1 as non-GC origin.

Using fresh samples, we estimated the frequency of IgV_H4-34 cases at 23%.

Within FR1, Q⁶W⁷ was unmutated in all 6 samples.

One sample had mutations in the A²⁴V²⁵Y²⁶ motif resulting in a change to A²⁴V²⁵F²⁶. The other 5 samples (83.3%) had unmutated AVY motifs.

We extracted genomic DNA from and performed next generation sequencing on the 5 samples with unmutated Q⁶W⁷ and A²⁴V²⁵Y²⁶ motifs using a customized capture library (SureSelectXT Target Enrichment System, Aqilent Technologies) covering genes involved in lymphomagenesis. The purified libraries were sequenced on the Illumina NextSeq500 platform at AGRF (Australian Genome Research Facility, Australia).

Several genes (FCGR3A,NOTCH2andNOTCH2NLR) had mutations in all 5 samples.FCGR3Ais an IgG Fc receptor gene, and mutations inFCGR3Ahave previously been linked to systemic lupus erythematosus (SLE).NOTCH2pathway genes are frequently mutated in DLBCL.CREBBPwas mutated in four of the five samples. Mutations inCREBBPhave previously been linked with DLBCL development and regulation of immune responses.

We identified high rates of IgV_H4-34 (23%) in our cohort of fresh samples as previously reported. Further, we noted preservation of the Q⁶W⁷ and A²⁴V²⁵Y²⁶ motifs in IgV_H4-34-expressing DLBCL. This over-representation of unmutated FR1 motifs suggests that the ability to recognise self-antigens likely provides important ongoing BCR signalling that promotes survival in DLBCL.

This study also highlights the difficulties in conducting DNA-based research on FFPE clinical samples which have not been collected for research purposes and the importance of tissue banking fresh samples.

Studies are currently being conducted into the efficacy of BCR pathway inhibitors e.g. ibrutinib in the treatment of DLBCL and testing for unmutated IgV_H4-34 FR1 motifs may present a method to predict patients who are more likely to respond.

Mutations in genes such as FCGR3A,NOTCH2andCREBBPmay work in conjunction with the preserved QW and AVY motifs to promote lymphomagenesis in IgV_H4-34-expressing B cells and may present targets for future research into treatment therapies.

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